The present invention relates generally to the field of medicine, and relates specifically to methods and compositions for modulating vascular permeability (VP).
Angiogenesis is a process of tissue vascularization that involves the growth of new developing blood vessels into a tissue, and is also referred to as neo-vascularization. The process is mediated by the infiltration of endothelial cells and smooth muscle cells. The process is believed to proceed in any one of three ways: the vessels can sprout from pre-existing vessels, de-novo development of vessels can arise from precursor cells (vasculogenesis), or existing small vessels can enlarge in diameter. Blood et al., Bioch. Biophys. Acta, 1032:89-118 (1990). For angiogenesis to occur, endothelial cells must first degrade and cross the blood vessel basement membrane in a similar manner used by tumor cells during invasion and metastasis formation. Angiogenesis is generally absent in adult or mature tissues, although it does occur in wound healing and in the corpus luteum growth cycle. See, for example, Moses et al., Science, 248:1408-1410 (1990).
While angiogenesis is an important process in neonatal growth, it is also important in wound healing and is a factor in the pathogenesis of a large variety of clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, macular degeneration by neovascularization of the retina, and like conditions. These clinical manifestations associated with angiogenesis are referred to as angiogenic diseases. Folkman et al., Science, 235:442-447 (1987).
It has been proposed that inhibition of angiogenesis would be a useful therapy for restricting tumor growth. Inhibition of angiogenesis has been proposed by (1) inhibition of release of xe2x80x9cangiogenic moleculesxe2x80x9d such as bFGF (basic fibroblast growth factor), (2) neutralization of angiogenic molecules, such as by use of anti-xcex2bFGF antibodies, (3) use of inhibitors of vitronectin receptor (xcex1vxcex23, and (4) inhibition of endothelial cell response to angiogenic stimuli. This latter strategy has received attention, Folkman et al., Cancer Biology, 3:89-96 (1992), have described several endothelial cell response inhibitors, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D3 analogs, alpha-interferon, and the like that might be used to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. No. 5,092,885, U.S. Pat. No. 5,112,946, U.S. Pat. No. 5,192,744, U.S. Pat. No. 5,202,352, U.S. Pat. No. 5,753,230 and U.S. Pat. No. 5,766,591. None of the inhibitors of angiogenesis described in the foregoing references involve the Src proteins, however.
It has been previously reported that angiogenesis depends on the interaction between vascular integrins and extracellular matrix proteins. Brooks et al., Science, 264:569-571 (1994). Furthermore, it was reported that programmed cell death (apoptosis) of angiogenic vascular cells is initiated by the interaction, which would be inhibited by certain antagonists of the vascular integrin xcex1vxcex23. Brooks et al., Cell, 79:1157-1164 (1994). More recently, it has been reported that the binding of matrix metalloproteinase-2 (MMP-2) to vitronectin receptor (xcex1vxcex25) can be inhibited using xcex1vxcex25 antagonists, and thereby inhibit the enzymatic function of the proteinase. Brooks et al., Cell, 85:683-693 (1996).
The brain vasculature is characterized by a highly restrictive blood-brain barrier that prohibits small molecules from extravasating into the surrounding brain tissue. The nature of the blood-brain barrier in mammals has been of special concern with pharmacological studies, as many drugs are routinely prevented from passing from the vasculature to the brain tissues because of the highly restrictive blood-brain barrier. The present invention involves the unexpected discovery that VP, as measured by vascular leakage of blood, can be modulated by src or yes. Moreover, VP has been associated with angiogenesis and other pathologies. Inflammation induced increased vascular permeability is associated with edema and swelling.
The present invention is directed to modulation of vascular permeability (VP) by tyrosine kinase Src, also referred to generically herein as Src, or the tyrosine kinase Yes, also referred to generically herein as Yes.
Thus, one aspect of the invention encompasses pharmaceutical compositions for modulating VP in target tissue of a mammal. The compositions of the invention comprise a therapeutically effective VP modulating amount of a mixture of tyrosine kinase protein Src and Yes, in a pharmaceutically acceptable carrier.
In compositions which comprise active Src and Yes kinase proteins, the expected modulation is a potentiation or increase in vascular permeability of the blood vessels in a target tissue. Where the desired Src protein is an active kinase, a preferred Src is Src-A. Another preferred active Src protein is one in which the amino acid residue at position 527 of the Src protein is any amino acid residue except for tyrosine, serine or threonine. The preferred active Yes protein will have the kinase activity of wild-type human Yes, such as that or the Yes-1 protein. Another preferred active Yes is one in which the kinase inactivating phosphorylation site of the Yes protein is mutated to abolish or minimize inactivating phosphorylation, similar to a mutation of amino acid residue 527 of Src to any amino acid residue except for tyrosine, serine or threonine.
Where the composition comprises Src and Yes protein that are inactive kinase proteins, the expected modulation is an inhibition or decrease in vascular permeability of the blood vessels in the target tissue. When the desired Src protein is an inactive protein, a preferred Src is Src 251. A further preferred inactive Src is Src K295M. A preferred inactive Yes protein will have diminished kinase activity as compared with the wild-type protein.
A further aspect of the claimed invention is a pharmaceutical composition comprising a therapeutically effective VP modulating amount of nucleic acid capable of expressing tyrosine kinase protein Src and Yes, when transfected into a target cell, in a suitable pharmaceutical carrier. The expressible nucleic acids encoding for Src or Yes protein can comprise nucleic acid segments which describe all or part of the Yes or Src protein. When transferred into target cells, the target cell transcribes and translates the nucleic acid sequence to express the desired protein.
Where the modulation is a potentiation or increase in vascular permeability of the blood vessels in the target tissue, Src encoding nucleic acid will encode active forms of Src, and Yes encoding nucleic acids will encode active forms of Yes kinase proteins. Once transferred into the target host cell, the nucleic acids will be expressed by the host cell. A preferred Src encoding nucleic acid encodes active Src A protein. A further preferred Src encoding nucleic acid encodes a mutated active Src where the amino acid residue at position 527 of the expressed Src protein is any amino acid residue except for tyrosine, serine or threonine. A preferred Yes encoding nucleic acid will encode the wild-type protein, or a protein modified to abolish or inhibit the inactivating phosphorylation site of the Yes protein, in a similar manner as the Src position 527 mutation described.
When the desired modulation is an inhibition or decrease in vascular permeability of the blood vessels in the target tissue, a preferred inactive Src encoding nucleic acid encodes Src 251 protein. A further preferred inactive Src encoding nucleic acid encodes inactive Src K295M. A preferred inactive Yes encoding nucleic acid will encode a protein that has diminished kinase activity.
It is envisioned that the compositions of the invention can comprise a mixture of nucleic acids, where each nucleic acid can comprise an expressible src or yes gene. In addition, it is envisioned that a single nucleic acid may comprise both a nucleic acid encoding for a Src protein, and a nucleic acid encoding for a Yes protein.
For refined modulation of angiogenesis and VP in target tissues, the pharmaceutical compositions of the invention can comprise a mixture of active or inactive tyrosine kinase protein Src, or tyrosine kinase protein Yes. Similarly, the pharmaceutical compositions of the invention can comprise a mixture of nucleic acid capable of expressing active or inactive tyrosine kinase protein Src, or tyrosine kinase protein Yes.
In this embodiment, by utilizing differentially expressible promoters or other such regulatory elements, a first low expressing first tyrosine kinase gene may be co-administered with a second high expressing second tyrosine kinase gene, according to the teaching of the invention. In this embodiment, an increase in angiogenesis can be accomplished while also maintaining, minimizing or reducing VP, by using a first low expressing active src gene, in combination with a second high expressing inactive yes gene. This co-administration can be accomplished by using separate expression vectors, or a single combined expression vector construct. Similarly, a decrease in angiogenesis can be accomplished while also maintaining, potentiating or increasing VP, by using a first low expressing inactive src gene, in combination with a second high expressing active yes gene. Further degrees of modulation can be accomplished by the various permutations of high/low and src/yes, in combination with selection of the activity of promotor elements, and inducible promoters.
It is envisioned that the individual src and yes genes may be under the regulatory control of the same or different regulatory nucleic acid sequences such as and not limited to enhancers, repressors, and promoter elements. When the two or more proteins are expressible from a single vector, it is envisioned that regulation and control of the transcription of the independent protein genes can be under the control of the same regulatory elements. It is also envisioned that regulation and control of transcription can be effected by two or more independently operating regulatory elements. Regulatory elements are known in the art, and can be constiutively active, or inducible, enhancer, promoter, suppressor, or the like, nucleic acid sequences.
It is envisioned that the nucleic acid compositions of the invention can comprise viral and/or non-viral gene transfer vector containing a nucleic acid segment encoding for a Src and/or Yes protein. Retroviral and non-viral gene transfer and expression vectors are known in the art, and described briefly below.
A preferred nucleic acid encodes Src-A protein. Another preferred active Src protein is one in which the amino acid residue at position 527 of the Src protein is any amino acid residue except for tyrosine, serine or threonine.
It is envisioned that a mixture of Src and Yes protein, and/or nucleic acid encoding for such protein, can combine active and inactive forms of protein, depending upon the level of modulation desired, and the coordinated effect on angiogenesis and VP desired, according to the teaching of the present invention.
A composition providing the Src or Yes protein can contain purified protein, biologically active fragments of natural protein, recombinantly produced Src or Yes protein or protein fragments or fusion proteins, or gene/nucleic acid expression vectors for expressing a Src or Yes protein, or mixtures thereof.
Where the Src or Yes protein is inactivated or inhibited, the modulation is an inhibition of VP. Where the Src or Yes protein is active or activated, the modulation is a potentiation of VP.
The present invention encompasses methods for treating mammalian tissue with a composition comprising a therapeutically effective, VP-modulating amount of a Src or Yes protein, or combination thereof. In the methods of the invention, Src and Yes tyrosine kinase protein, or nucleic acid expression vectors capable of expressing such protein is administered to tissue suffering from a disease condition that responds to modulation of VP.
Where the therapeuticly effective VP modulating effect desired is an increase or potentiation of VP, it is contemplated that active forms of Src protein and/or Yes protein can be administered. Similarly, the methods encompass the administration of expressible nucleic acids which encode active or inactive forms of Src protein and/or Yes protein, accordingly.
The tissue to be treated can be any tissue in which modulation of VP is desirable. Therapeutic treatment is accomplished by contacting the target tissue with an effective amount of the desired modulating composition, and allowed sufficient time of contact for the protein or nucleic acid components of the pharmaceutical to enter the target tissue. For VP inhibition, it is useful to treat diseased tissue where deleterious vascular leaking is occurring. Exemplary tissues include inflamed tissue, tissues associated with stroke, myocardial infarction, or other blockage of normal flow, tissues undergoing restenosis, and the like tissues.
For potentiation, it is useful to treat patients with ischemic limbs in which there is poor circulation in the limbs from diabetic or other conditions, or for potentiating the administration of drugs to the brain across the blood-brain barrier. Patients with chronic wounds which do not heal and therefore could benefit from the increase in vascular cell proliferation and neovascularization as modulated by VP can be treated as well.
A further aspect of the present invention are articles of manufacture which comprise packaging material and a pharmaceutical composition contained within said packaging material, wherein said pharmaceutical composition is capable of modulating vascular permeability in a tissue suffering from a disease condition, wherein said packaging material comprises a label which indicates that said pharmaceutical composition can be used for treating disease conditions by modulating vascular permeability, and wherein said pharmaceutical composition comprises a therapeutically effective amount of tyrosine kinase protein Yes, in a pharmaceutically acceptable carrier. This embodiment encompasses Yes protein in active or inactive form, and also nucleic acids encoding for active or inactive Yes protein. Both retroviral and non-viral gene transfer/expression vectors can contain a nucleic acid segment encoding for Yes protein, either in active or inactive form, or both. When both active and inactive forms of a protein kinase gene are present, it is contemplated that the genes are under separate inducible promoter regulation to allow for alternative expression, as desired.
A further aspect of the present invention are articles of manufacture wherein the pharmaceutical composition comprises a therapeutically effective VP modulating amount of a tyrosine kinase protein Src and Yes, in a pharmaceutically acceptable carrier. Where the article of manufacture is packaged to indicate a potentiating VP modulating effect, Src and Yes are in active form. A preferred active Src is Src-A protein. Another preferred active Src protein is one in which the amino acid residue at position 527 of the Src protein is any amino acid residue except for tyrosine, serine or threonine.
A further aspect of the present invention are articles of manufacture which comprise a pharmaceutical composition wherein said pharmaceutical composition comprises a therapeutically effective VP modulating amount of an inactive tyrosine kinase protein Src and Yes protein, in a pharmaceutically acceptable carrier, where the desired modulation is an inactivation or inhibition of VP. A preferred inactive Src is Src 251 protein. Another preferred inactive Src protein is Src K295M.
Similarly, a further aspect of the present invention are articles of manufacture wherein the pharmaceutical composition comprises a nucleic acid capable of expressing tyrosine kinase protein Src and Yes, in a suitable pharmaceutical carrier. A preferred nucleic acid component of the pharmaceutical composition of this article of manufacture encode an active Src protein, where the modulation desired is a potentiation or activation of VP. Further envisioned are nucleic acid encoding active Yes protein. A preferred active Src is Src-A protein. Another preferred active Src encoding nucleic acid is one in which the amino acid residue at position 527 of the Src protein is any amino acid residue except for tyrosine, serine or threonine. It is also envisioned that a single nucleic acid can be constructed which will express both yes and src, either independently regulated, or under transcriptional control of the same promoter, enhancer, suppressor, repressor or other suitable regulatory nucleic acid sequence.
The pharmaceutical composition of the article of manufacture can vary depending upon the desired modulatory effect, and the packaging labeling will correspondingly vary as well.